Cell culture of mammalian cells has long been used for the production of many vaccines and genetically engineered proteins. Animal cells are generally categorized according to their anchorage-dependence. Some cell types, such as lymphocytes, can grow in suspension; others, called "anchorage-dependent", including fibroblasts and epithelial and endothelial cells, must attach to a surface and spread out in order to grow. Other cells can grow either in suspension or anchored to a surface.
Anchorage-dependent cells have historically been cultivated on the walls of roller bottles or non-agitated vessels such as tissue culture flasks, which are used in many laboratories. As the necessity has developed to provide large amounts of certain antiviral vaccines, genetically engineered proteins, and other cell-derived products, attempts have been made to develop new systems for larger scale production of cells.
The first focus of this development effort was to increase the growth surface area per unit vessel volume and to implement convenient and appropriate environmental controls. Some of these technologies involved the use of packed-glass beads, stacked plates, rotating multiple tubes, and roller bottles with spiral films inside.
Among the most important advances in the field of cell culture technology have been the use of microcarriers and more recently, the use of hollow fiber systems. Original microcarriers developed by van Wezel (van Wezel, A. L., "Growth of Cell-Strains and Primary Cells on Micro-carders in Homogeneous Culture," Nature 216:64-65 (1967)) consisted of positively charged DEAE-dextran beads suspended in culture media in a stirred vessel. Cells would attach to the bead surface and grow as a monolayer.
Hollow fiber bioreactor configurations serve to compartmentalize the bioreactors. In one common configuration, these units allow cells to grow on the outside surfaces of bundles of parallel fibers enclosed in an outer compartment. Nutrient- and gas-enriched medium flows through the fibers' hollow centers. Cell products are concentrated in the outer compartment of the bioreactor because the inner surface of the fiber includes an ultrafiltration membrane that excludes large molecular-weight cell products.
Bioreactors have certain minimum requirements: an aeration system is required to bring the correct amount of oxygen to the cells without causing shear damage; surfaces are required for supporting anchorage-dependent cells; and means are required to enable operators to sample and monitor the contents of the bioreactor without contaminating the culture.
The various bioreactors have encountered similar problems in culturing cells. With respect to anchorage-dependent cells, specific cell culture parameters in need of improvement include: (1) better initial attachment and growth of cells to decrease the concentration of cells required for inoculation of a culture; (2) improved long-term cell adhesion, viability, and productivity to increase the useful life of the bioreactor; and (3) alteration of growth conditions to allow lower concentrations of serum to be used in the culture medium.
The adhesion of cells to a surface is a multi-step process, consisting of initial attachment (characterized by weak binding and little cell shape change) followed by cell spreading (which produces stronger binding of cells to the substrate) (Grinnell, F., "Cellular Adhesiveness and Extracellular Substrata", Internat. Rev. Cytology 53:65-144 (1978)). The initial attachment can be mediated by non-specific mechanisms such as charged surfaces (Grinnell, F., "Cellular Adhesiveness and Extracellular Substrata", Internat. Rev. Cytology 53:65-144 (1978) and Microcarrier Cell Culture. Principles and Methods, Pharmacia Fine Chemicals, Uppsala, Sweden, pages 5-33 (1981)). In contrast to initial attachment, cell spreading seems to require the presence of specific receptor-ligand interactions between cell surface receptors and certain cell adhesion glycoproteins, such as fibronectin, laminin, and collagens (Kleinman, H. K., Luckenbill-Edds, F. W. Cannon, and G. C. Sephel, "Use of Extracellular Matrix Components for Cell Culture", Anal. Biochem. 166:1-13 (1987)). All three types of these glycoproteins have been purified and added to tissue culture surfaces to promote cell adhesion and cell growth (Kleinman, H. K., Luckenbill-Edds, F. W. Cannon, and G. C. Sephel, "Use of Extracellular Matrix Components for Cell Culture", Anal. Biochem. 166:1-13 (1987)). Studies have shown that a coating of gelatin or denatured collagen on microcarders facilitates the attachment and growth of mammalian cells (Microcarrier Cell Culture. Principles and Methods, Pharmacia Fine Chemicals, Uppsala, Sweden, pages 5-33 (1981)).
Early microcarriers were in the form of DEAE-derivatized dextran beads. The use of these beads, however, produced certain deleterious effects. For example, a high initial cell death rate and inadequate cell growth was observed with cells attached to beads that contain an ion-exchange capacity that was too high. Two methods that have been proposed to overcome some of these deleterious effects involved (1) attaching a lower density of positively-charged molecules to the beads, in order to provide a charge capacity of 0.1-4.5 meg/g dextran (see, e.g., U.S. Pat. No. 4,293,654), and (2) adsorbing polyanions onto the positively-charged microcarriers, in order to neutralize some of the excess charge (see, e.g., U.S. Pat. No. 4,036,693).
It has been reported that the adsorption of an attachment glycoprotein (fibronectin) from serum onto the surface of positively-charged microcarriers promotes cell spreading in non-agitated cultures (Lai, C-S, E. G. Ankel and L. E. Hopwood, "Membrane Fluidity of Chinese Hamster Ovary Cells on Plasma Fibronectin-Coated Microcarriers", Exp. Cell Res. 150:77-83 (1984); Microcarder Cell Culture. Principles and Methods, Pharmacia Fine Chemicals, Uppsala, Sweden, pages 5-33 (1981)). On the other hand, the presence of adsorbed fibronectin has been shown to have the undesirable effect of decreasing the rate of cell attachment to stirred, i.e., agitated, microcarders (Himes, V. B. and W. S. Hu, "Attachment and Growth of Mammalian Cells on Microcarders with Different Ion Exchange Capacities", Biotech. Bioeng. 29:1155-1163 (1987)).
Cell adhesion proteins (e.g., fibronectin, laminin, and collagens) used in the absence of positively-charged groups have worked well to promote the growth and spreading of cells in non-agitated cell culture devices, but do not appear to effectively attract and attach cells with a sufficient rate or tenacity in agitated devices.
The incorporation of positive charges onto macroporous gelatin microcarriers was reported to greatly improve the rate of cell attachment to these microcarriers (Kim, J-H, H-S Lim, B-K Han, M. V. Peshwa, and W. S. Hu, "Characterization of Cell Growth and Improvement of Attachment Kinetics on Macroporous Microcarriers", presented at the Fourth Annual Meeting of the Japanese Association for Animal Cell Technology, Fukuoka, Japan (November 1991)).
Most currently used microcarriers use porous non-rigid dextran as a support matrix. This compressible matrix is believed by some to reduce the potential for damage to the microcarriers and attached cells when the microcarriers collide in agitated reactors Microcarder Cell Culture. Principles and Methods, Pharmacia Fine Chemicals, Uppsala, Sweden, pages 5-33 (1981)). Such porous microcarriers, however, frequently also have the disadvantage of retaining cellular products that are secreted into the medium (thus complicating the harvesting of desired cell products) as well as the disadvantage of adsorbing growth factors and other serum components, thus reducing their levels in the culture media (Butler, M., "Growth Limitations in Microcarder Cultures", Adv. Biochem. Eng./Biotech. 4:57-84 (1987)).
Polystyrene microcarriers produce superior cell growth, with higher recovery of products; however, currently available polystyrene microcarders produce unacceptably low rates of cell attachment.
While not considered to be art against the instant application, PCT application publication No. WO 91/07485, published May 30, 1991 (now abandoned), which corresponded to U.S. Ser. No. 434,092 (assigned to the assignee of the instant application) describes, inter alia, a bioreactor cell culture surface having a cell adhesion factor and positively charged chemical moiety.
In spite of the art and other earlier efforts described above, those involved in the cell culture of anchorage-dependent cells remain desirous of a bioreactor support surface having both cell adhesion factor and positive charge provided in a manner that is stable in the course of agitation during incubation.